The coding region of GJB2 was assessed via Sanger sequencing technique using a DNA sequencer (Applied Biosystems, USA). NCBI’s application was utilized to compare reference genome with the obtained sequence of GJB2. WES data were used for the control group. Exome capture was then carried out. The TruSeq® DNA Sample Prep Kit v2-Set A (Illumina) was used to prepare a genomic library. Q30 standard was utilized to filter the reads. Burrows-Wheeler aligner (BWA) software was utilized for aligning. Polymerase chain reaction (PCR) was performed for amplifying the amplicons of the GJB2 gene. The obtained hybridized mixture having specific tag probes was then affixed on to the microarray chip. The chip signals were then analyzed by scanning and imaging using the LuxScan Microarray Scanner and detection system. GJB2-Exon2- F: 5′-TTGGTGTTTGCTCAGGAAGA-3′ GJB2-Exon2- R: 5′-GGCCTACAGGGGTTTCAAAT-3′. Procedures used were: 1) UNG treatment at 50°C for 2 min, 2) Pre-denaturation at 95°C for 10 min, and 3) Drop cycle program 95°C, 15 s; 65°C, 15 s (1°C drop per cycle); 76°C, 20 s (10 cycles). The melting curve analysis program was 95°C for 1 min, 35°C for 3 min, and 40°C to 80°C, set to collect channel fluorescence signal at this stage.
Truseq dna sample prep kit v2 set a
Manufactured by Illumina
Sourced in United States
The TruSeq® DNA Sample Prep Kit v2-Set A is a laboratory equipment product designed for DNA sample preparation. It provides the necessary reagents and protocols for sample preparation prior to sequencing. The kit includes reagents for fragmentation, end-repair, A-tailing, and adapter ligation of DNA samples.
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Lab products found in correlation
Truseq pe cluster kit v3, by Illumina (2 mentions)
Truseq sbs v3 chemistry, by Illumina (2 mentions)
Casava 1, by Illumina (2 mentions)
Truseq dna sample preparation v2, by Illumina (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 instrument, by Illumina (1 mentions)
Phusion polymerase, by Thermo Fisher Scientific (1 mentions)
D1000 screentape, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Minelute gel extraction kit, by Qiagen (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
5 protocols using truseq dna sample prep kit v2 set a
1
GJB2 Gene Sequencing and Analysis
2
Illumina TruSeq DNA Library Preparation
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Sequencing libraries were prepared using the TruSeq DNA Sample Prep kit v2-Set A (Illumina #FC-121-2001) for blunting, adding “A” overhangs, and adapter ligation. Excess adapters were removed by using AMPure XP beads (Beckman Coulter #A63880) and DNA was eluted in 38 μl 10 mM Tris-HCl pH 8.5 (EB from Qiagen #19086). Libraries were amplified with PCR (17 cycles—30 s at 98 °C, 30 s at 60 °C, 30 s at 72 °C) using Phusion polymerase (ThermoFisher Scientific #F530-S) and primers complementary to the adapters. The DNA was cleaned up using the MinElute PCR Purification kit (Qiagen #28006) and eluted with 11 μl EB. Finally, 300–600 bp fragments were purified from 1.5% agarose gel (1xTAE buffer) using the MinElute Gel Extraction kit (Qiagen #28606). Library concentration was determined by Qubit (Invitrogen) using the dsDNA HS Assay Kit (Invitrogen #Q32851), and library quality and size range were determined by TapeStation (Agilent) using High Sensitivity D1000 ScreenTape (Agilent #5067-5584). Pooled library (18 total—nine from DNA and 9 from total RNA—see below) concentration was determined using a KAPA library quantification kit (Roche #07960140001). Libraries were sequenced on an Illumina NextSeq 500 instrument (2 × 150 bp paired-end reads, HighOutput mode v2.5).
3
Whole Blood DNA Methylation Analysis
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Whole blood samples were obtained from eight smoking and eight non‐smoking mother–child pairs (see Appendix Supplementary Methods and Fig EV1 ). Libraries were prepared using the TruSeq DNA Sample Prep Kit v2‐Set A (Illumina Inc., San Diego, CA, USA) according to the manufacturer's instructions. Adapter‐ligated libraries were treated with bisulfite and PCR‐amplified (see Appendix Supplementary Methods and Table EV10 ). Sequencing on HiSeq2000 (101‐bp paired‐end) was performed using standard Illumina protocols and the 200‐cycle TruSeq SBS Kit v3 (Illumina Inc., San Diego, CA, USA).
4
Whole Genome Sequencing and Copy Number Analysis
5
Whole Genome Sequencing and Copy Number Analysis
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Sequencing libraries were prepared according to the TruSeq DNA v2 sample preparation EUC #15026489 revA using reagents from the TruSeq DNA v2 sample prep kit set A and set B (Illumina). A 14 pM solution of 5-6 DNA libraries pooled in equimolar amounts was subjected to cluster generation on the cBot instrument (Illumina Inc.) using the TruSeq PE cluster kit v3. Paired-end sequencing was performed for 100 cycles using a HiSeq2000 instrument (Illumina Inc.) using TruSeq SBS chemistry v3, according to the manufacturer’s protocols. Base calling was done on the instrument by RTA 1.13.48 and the resulting bcl files were filtered, demultiplexed, allowing for one mismatch base, and converted to fastq format with tools provided by CASAVA-1.8 (Illumina Inc.). Copy number profiles were computed from NGS-data using the Control-FREEC software40 (link). The sequence read depth was used to calculate an equivalent to the Log R Ratio using a sliding window approach. We applied a fixed window size of 5 kb and the other settings were default. The NGS dataset was composed of 100 whole genomes with 5x average coverage and additional exomes, with an average coverage of 17x.
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